Genetic polymorphisms in the human HMG-CoA reductase gene and their use in diagnosis and treatment of diseases

ABSTRACT

This invention relates to polymorphisms in the human HMG-CoA reductase gene and corresponding novel allelic polypeptides encoded thereby. Particular polymorphisms are described in the promoter, exon 15 and introns 2, 5, 15 and 18. The invention also relates to methods and materials for analysing allelic variation in the HMG CoA reductase gene, and to the use of HMG-CoA reductase polymorphism in the diagnosis and treatment of HMG-CoA reductase mediated diseases such as dyslipidemia and other cardiovascular diseases such as myocardial infarction and stroke.

[0001] This invention relates to polymorphisms in the human HMG-CoAreductase gene and corresponding novel allelic polypeptides encodedthereby. The invention also relates to methods and materials foranalysing allelic variation in the HMG CoA reductase gene, and to theuse of HMG-CoA reductase polymorphism in the diagnosis and treatment ofHMG-CoA reductase mediated diseases such as dyslipidemia and othercardiovascular diseases such as myocardial infarction and stroke.

[0002] At the time of priority filing, there were no known polymorphismsin the HMG-CoA reductase gene. On Oct. 7, 1999, in PCT Application WO99/50454, Lander et al published on a Ile to Val polymorphism atposition 638 (see FIG. 1B therein).

[0003] In the human HMG CoA reductase gene a single donor splice site isused to excise the intron in the 5′ untranslated region. There aremultiple mRNAs due to alternative start sites, all of which have shortuntranslated regions of 68 to 100 nucleotides (“Conservation of promotersequence but not complex intron splicing pattern in human and hamstergenes for 3-hydroxy-3-methylglutaryl coenzyme A reductase”; Mol. Cell.Biol. 7:1881-1893(1987).)

[0004] The HMG-CoA reductase gene has been cloned as cDNA and publishedas EMBL Accession number M11058 (2904 bp) as defined by SEQ ID NO 44.All positions herein of polymorphisms in the coding sequence relate tothe position in SEQ ID NO 44 unless stated otherwise or apparent fromthe context. The protein sequence of the HMG-CoA reductase has also beenbeen published in Luskey K. L. et al “Human 3-hydroxy-3-methylglutarylcoenzyme A reductase. Conserved domains responsible for catalyticactivity and sterol-regulated degradation”; J. Biol. Chem.260:10271-10277(1985).

[0005] A partial genomic sequence of HMG-CoA reductase, including thepromoter and exon-1, is published as EMBL Accession number M15959 (1227bp) as defined by SEQ ID NO 45 herein. All positions herein ofpolymorphisms in the promoter region relate to the position in SEQ ID NO45 unless stated otherwise or apparent from the context.

[0006] All positions herein of polymorphisms in the intron regionsrelate to the position of the relevant intron sequence disclosed hereinunless stated otherwise or apparent from the context.

[0007] HMG-CoA reductase is the rate-limiting enzyme for cholesterolsynthesis and is regulated via a negative feedback mechanism mediated bysterols and non-sterol metabolites derived from mevalonate, the productof the reaction catalyzed by reductase. Normally in mammalian cells,this enzyme is suppressed by cholesterol derived from theinternalization and degradation of LDL via the LDL receptor. Competitiveinhibitors (termed “statins”) of the reductase induce the expression ofLDL receptors in the liver, which in turn increases the catabolism ofplasma LDL and lowers the plasma concentration of cholesterol, animportant determinant of atherosclerosis.

[0008] The sequence coding for the highly conserved membrane boundregion of the protein is located at positions 51-1067, that coding forthe linker part of the protein at positions 1068-1397 and for thestrongly conserved water-soluble catalytic part at positions 1398-2714.

[0009] One approach is to use knowledge of polymorphisms to helpidentify patients most suited to therapy with particular pharmaceuticalagents (this is often termed “pharmacogenetics”). Pharmacogenetics canalso be used in pharmaceutical research to assist the drug selectionprocess. Polymorphisms are used in mapping the human genome and toelucidate the genetic component of diseases. The reader is directed tothe following references for background details on pharmacogenetics andother uses of polymorphism detection: Linder et al. (1997), ClinicalChemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249;International Patent Application WO 97/40462, Spectra Biomedical; andSchafer et al. (1998), Nature Biotechnology, 16, 33.

[0010] Clinical trials have shown that patient response to treatmentwith pharmaceuticals is often heterogeneous. Thus there is a need forimproved approaches to pharmaceutical agent design and therapy.

[0011] Point mutations in polypeptides will be referred to as follows:natural amino acid (using 1 or 3 letter nomenclature), position, newamino acid. For (a hypothetical) example “D25K” or “Asp25Lys” means thatat position 25 an aspartic acid (D) has been changed to lysine (K).Multiple mutations in one polypeptide will be shown between squarebrackets with individual mutations separated by commas.

[0012] The present invention is based on the discovery of the genomicstructure of HMG-CoA reductase and polymorphism therein. In particular,we have found one single nucleotide polymorphism (SNP) in the codingsequence of the HMG-CoA reductase gene, 2 SNPs in the promoter sequenceof the HMG-CoA reductase gene and 5 SNPs in the intron sequence of theHMG-CoA reductase gene as well as the genomic structure of the gene andnovel sequence allowing the discovery of SNPs in the exons and intronsof the gene.

[0013] According to one aspect of the present invention there isprovided a method for the diagnosis of a single nucleotide polymorphismin HMG-CoA reductase in a human, which method comprises determining thesequence of the nucleic acid of the human at at least one polymorphicposition and determining the status of the human by reference topolymorphism in the HMG-CoA reductase gene. Preferred polymorphicpositions are one or more of the following positions:

[0014] position 1962 in the coding sequence of the HMG-CoA reductasegene as defined by the position in SEQ ID NO: 44, and/or

[0015] positions 46 or 267 in the promoter sequence of the HMG-CoAreductase gene as defined by the positions in SEQ ID NO: 45; and/or

[0016] position 129 in intron 2 as defined by the position in SEQ IDNO:20,

[0017] position 550 in intron 5 as defined by the position in SEQ ID NO:24,

[0018] position 37 in intron 15 as defined by the position in SEQ IDNO:37, or

[0019] position 345 in intron 18 as defined by the position in SEQ IDNO:40 of the HMG-CoA reductase gene.

[0020] According to another aspect of the present invention there isprovided a method for the diagnosis of a single nucleotide polymorphismin HMG-CoA reductase in a human, which method comprises determining thesequence of the nucleic acid of the human at at least one polymorphicposition and determining the status of the human by reference topolymorphism in the HMG-CoA reductase gene. Preferred polymorphicpositions are one or more of the following positions:

[0021] position 1962 in the coding sequence of the HMG-CoA reductasegene as defined by the position in SEQ ID NO: 44, and/or

[0022] positions 46 or 267 in the promoter sequence of the HMG-CoAreductase gene as defined by the positions in SEQ ID NO: 45; and/or

[0023] position 129 in intron 2 as defined by the position in SEQ IDNO:20,

[0024] position 550 in intron 5 as defined by the position in SEQ ID NO:24,

[0025] position 558 in intron 14 as defined by the position in SEQ IDNO:36, or position 345 in intron 18 as defined by the position in SEQ IDNO:40 of the HMG-CoA reductase gene.

[0026] The term human includes both a human having or suspected ofhaving a HMG-CoA reductase mediated disease and an asymptomatic humanwho may be tested for predisposition or susceptibility to such disease.At each position the human may be homozygous for an allele or the humanmay be a heterozygote.

[0027] The term single nucleotide polymorphism includes singlenucleotide substitution, nucleotide insertion and nucleotide deletionwhich in the case of insertion and deletion includes insertion ordeletion of one or more nucleotides at a position of a gene.

[0028] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 1962 of the coding sequence is presence of Aand/or G.

[0029] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 46 of the promoter is presence of T and/or C.

[0030] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 267 of the promoter is presence of C and/or G.

[0031] In another embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 129 of intron 2 is the presence or absence ofan insertion of AA.

[0032] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 550 of intron 5 is presence of T and/or A.

[0033] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 37 of intron 15 is presence of A and/or G.

[0034] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the single nucleotidepolymorphism at position 345 of intron 18 is presence of T and/or C.

[0035] The method for diagnosis is preferably one in which the sequenceis determined by a method selected from amplification refractorymutation system and restriction fragment length polymorphism.

[0036] Allelic variation at position 1962 consists of a single basesubstitution from A (the published base), preferably to G.

[0037] Allelic variation at position 46 consists of a single basesubstitution from C (the published case), preferably to G.

[0038] Allelic variation at position 267 consists of a single basesubstitution from T (the published base), preferably to C.

[0039] Allelic variation at position 129 consists of a presence orabsence of insertion, preferably to presence or absence of the insertionof AA.

[0040] Allelic variation at position 550 consists of a single basesubstitution from T, preferably to A.

[0041] Allelic variation at position 37 consists of a single basesubstitution from A, preferably to G.

[0042] Allelic variation at position 345 consists of a single basesubstitution from T, preferably to C.

[0043] The status of the individual may be determined by reference toallelic variation at any one, two, three, four, five, six or seven ormore positions.

[0044] The test sample of nucleic acid is conveniently a sample ofblood, bronchoalveolar lavage fluid, sputum, or other body fluid ortissue obtained from an individual. It will be appreciated that the testsample may equally be a nucleic acid sequence corresponding to thesequence in the test sample, that is to say that all or a part of theregion in the sample nucleic acid may firstly be amplified using anyconvenient technique e.g. PCR, before analysis of allelic variation.

[0045] It will be apparent to the person skilled in the art that thereare a large number of analytical procedures which may be used to detectthe presence or absence of variant nucleotides at one or morepolymorphic positions of the invention. In general, the detection ofallelic variation requires a mutation discrimination technique,optionally an amplification reaction and optionally a signal generationsystem. Table 1 lists a number of mutation detection techniques, somebased on the PCR. These may be used in combination with a number ofsignal generation systems, a selection of which is listed in Table 2.Further amplification techniques are listed in Table 3. Many currentmethods for the detection of allelic variation are reviewed by Nollau etal., Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, forexample “Laboratory Protocols for Mutation Detection”, Ed. by U.Landegren, Oxford University Press, 1996 and “PCR”, 2^(nd) Edition byNewton & Graham, BIOS Scientific Publishers Limited, 1997. ALEX ™Amplification refractory mutation system linear extension APEX Arrayedprimer extension ARMS ™ Amplification refractory mutation system b-DNABranched DNA bp base pair CMC Chemical mismatch cleavage COPSCompetitive oligonucleotide priming system DGGE Denaturing gradient gelelectrophoresis FRET Fluorescence resonance energy transfer HDL highdensity lipoprotein HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A LCRLigase chain reaction LDL low density lipoprotein MASDA Multiple allelespecific diagnostic assay NASBA Nucleic acid sequence basedamplification OLA Oligonucleotide ligation assay PCR Polymerase chainreaction PTT Protein truncation test RFLP Restriction fragment lengthpolymorphism SDA Strand displacement amplification SNP Single nucleotidepolymorphism SSCP Single-strand conformation polymorphism analysis SSRSelf sustained replication TGGE Temperature gradient gel electrophoresis

[0046] TABLE 1 Mutation Detection Techniques General: DNA sequencing,Sequencing by hybridisation Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase,Heteroduplex analysis, CMC, Enzymatic mismatch cleavage * Note: notuseful for detection of promoter polymorphisms. Hybridisation BasedSolid phase hybridisation: Dot blots,  MASDA, Reverse dot blots,Oligonucleotide arrays (DNA Chips) Solution phasehybridisation: Taqman ™ - US-5210015 & US-5487972 (Hoffmann-La Roche),Molecular Beacons - Tyagi et al (1996), Nature Biotechnology, 14, 303;WO 95/13399 (Public Health Inst., New York) Extension Based: ARMS ™,ALEX ™ - European Patent No. EP 332435 B1 (Zeneca Limited), COPS - Gibbset al (1989), Nucleic Acids Research, 17, 2347. Incorporation Based:Mini-sequencing, APEX Restriction Enzyme Based: RFLP, Restriction sitegenerating PCR Ligation Based: OLA Other: Invader assay

[0047] TABLE 2 Signal Generation or Detection Systems Fluorescence:FRET, Fluorescence quenching, Fluorescence polarisation - United KingdomPatent No. 2228998 (Zeneca Limited) Other: Chemiluminescence,Electrochemiluminescence, Raman, Radioactivity, Colorimetric,Hybridisation protection assay, Mass spectrometry

[0048] TABLE 3 Further Amplification Methods SSR, NASBA, LCR, SDA, b-DNA

[0049] Preferred mutation detection techniques include ARMS™, ALEX™,COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCRand FRET techniques.

[0050] Particularly preferred methods include ARMS™ and RFLP basedmethods. ARMS™ is an especially preferred method.

[0051] In a further aspect, the diagnostic methods of the invention areused to assess the pharmacogenetics of therapeutic compounds in thetreatment of HMG-CoA reductase mediated diseases.

[0052] Assays, for example reporter-based assays, may be devised todetect whether one or more of the above polymorphisms affecttranscription levels and/or message stability.

[0053] Individuals who carry particular allelic variants of the HMG-CoAreductase gene may therefore exhibit differences in their ability toregulate protein biosynthesis under different physiological conditionsand will display altered abilities to react to different diseases. Inaddition, differences in protein regulation arising as a result ofallelic variation may have a direct effect on the response of anindividual to drug therapy. The diagnostic methods of the invention maybe useful both to predict the clinical response to such agents and todetermine therapeutic dose.

[0054] In a further aspect, the diagnostic methods of the invention, areused to assess the predisposition and/or susceptibility of an individualto diseases mediated by HMG-CoA reductase. This may be particularlyrelevant in the development of hyperlipoproteinemia and cardiovasculardisease and the present invention may be used to recognise individualswho are particularly at risk from developing these conditions.

[0055] In a further aspect, the diagnostic methods of the invention areused in the development of new drug therapies which selectively targetone or more allelic variants of the HMG-CoA reductase gene.Identification of a link between a particular allelic variant andpredisposition to disease development or response to drug therapy mayhave a significant impact on the design of new drugs. Drugs may bedesigned to regulate the biological activity of variants implicated inthe disease process whilst minimising effects on other variants.

[0056] In a further diagnostic aspect of the invention the presence orabsence of variant nucleotides is detected by reference to the loss orgain of, optionally engineered, sites recognised by restriction enzymes.

[0057] According to another aspect of the present invention there isprovided a human HMG-CoA reductase gene or its complementary strandcomprising a polymorphism, preferably corresponding with one or more ofpositions defined herein or a fragment thereof of at least 20 basescomprising at least one polymorphism.

[0058] Fragments are at least 17 bases, more preferably at least 20bases, more preferably at least 30 bases.

[0059] According to another aspect of the present invention there isprovided a polynucleotide comprising at least 20 bases of the humanHMG-CoA reductase gene and comprising a polymorphism selected from anyone of the following: Region SEQ ID Position Polymorphism Exon 15 SEQ IDNO: 44 1962 A → G promoter SEQ ID NO: 45 46 C → G promoter SEQ ID NO: 45267 T → C Intron 2 SEQ ID NO: 20 129 CT → CAAT Intron 5 SEQ ID NO: 24550 T → A Intron 15 SEQ ID NO: 37 37 A → G Intron 18 SEQ ID NO: 40 345 T→ C

[0060] In another embodiment the following polymorphisms are preferred:Region SEQ ID Position Polymorphism promoter SEQ ID NO: 45 46 C → Gpromoter SEQ ID NO: 45 267 T → C Intron 2 SEQ ID NO: 20 129 CT → CAATIntron 5 SEQ ID NO: 24 550 T → A Intron 15 SEQ ID NO: 37 37 A → G Intron18 SEQ ID NO: 40 345 T → C

[0061] According to another aspect of the present invention there isprovided a human HMG-CoA reductase gene or its complementary strandcomprising a polymorphism, preferably corresponding with one or more thepositions defined herein or a fragment thereof of at least 20 basescomprising at least one polymorphism.

[0062] Fragments are at least 17 bases, more preferably at least 20bases, more preferably at least 30 bases.

[0063] The invention further provides a nucleotide primer which candetect a polymorphism of the invention.

[0064] According to another aspect of the present invention there isprovided an allele specific primer capable of detecting a HMG-CoAreductase gene polymorphism, preferably at one or more of the positionsas defined herein.

[0065] An allele specific primer is used, generally together with aconstant primer, in an amplification reaction such as a PCR reaction,which provides the discrimination between alleles through selectiveamplification of one allele at a particular sequence position e.g. asused for ARMS™ assays. The allele specific primer is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

[0066] An allele specific primer preferably corresponds exactly with theallele to be detected but derivatives thereof are also contemplatedwherein about 6-8 of the nucleotides at the 3′ terminus correspond withthe allele to be detected and wherein up to 10, such as up to 8, 6, 4,2, or 1 of the remaining nucleotides may be varied without significantlyaffecting the properties of the primer.

[0067] Primers may be manufactured using any convenient method ofsynthesis. Examples of such methods may be found in standard textbooks,for example “Protocols for Oligonucleotides and Analogues; Synthesis andProperties,” Methods in Molecular Biology Series; Volume 20; Ed. SudhirAgrawal, Humana ISBN: 0-89603-247-7; 1993; 1^(st) Edition. If requiredthe primer(s) may be labelled to facilitate detection.

[0068] According to another aspect of the present invention there isprovided an allele-specific oligonucleotide probe capable of detecting aHMG-CoA reductase gene polymorphism, preferably at one or more of thepositions defined herein.

[0069] The allele-specific oligonucleotide probe is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

[0070] The design of such probes will be apparent to the molecularbiologist of ordinary skill. Such probes are of any convenient lengthsuch as up to 50 bases, up to 40 bases, more conveniently up to 30 basesin length, such as for example 8-25 or 8-15 bases in length. In generalsuch probes will comprise base sequences entirely complementary to thecorresponding wild type or variant locus in the gene. However, ifrequired one or more mismatches may be introduced, provided that thediscriminatory power of the oligonucleotide probe is not undulyaffected. The probes of the invention may carry one or more labels tofacilitate detection.

[0071] According to another aspect of the present invention there isprovided an allele specific primer or an allele specific oligonucleotideprobe capable of detecting a HMG-CoA reductase gene polymorphism at oneof the positions defined herein.

[0072] According to another aspect of the present invention there isprovided a diagnostic kit comprising an allele specific oligonucleotideprobe of the invention and/or an allele-specific primer of theinvention.

[0073] The diagnostic kits may comprise appropriate packaging andinstructions for use in the methods of the invention. Such kits mayfurther comprise appropriate buffer(s) and polymerase(s) such asthermostable polymerases, for example taq polymerase.

[0074] In another aspect of the invention, the single nucleotidepolymorphisms of this invention may be used as genetic markers inlinkage studies. This particularly applies to the polymorphisms ofrelatively high frequency in introns 5 and 18 (see below). The HMG-CoAreductase gene has been mapped to chromosome 5q13.3-q14 (Luskey K. L.,Stevens B.; RT “Human 3-hydroxy-3-methylglutaryl coenzyme A reductase.Conserved domains responsible for catalytic activity andsterol-regulated degradation”; J. Biol. Chem. 260:10271-10277 (1985)).Low frequency polymorphisms may be particularly useful for haplotypingas described below. A haplotype is a set of alleles found at linkedpolymorphic sites (such as within a gene) on a single (paternal ormaternal) chromosome. If recombination within the gene is random, theremay be as many as 2^(n) haplotypes, where 2 is the number of alleles ateach SNP and n is the number of SNPs. One approach to identifyingmutations or polymorphisms which are correlated with clinical responseis to carry out an association study using all the haplotypes that canbe identified in the population of interest. The frequency of eachhaplotype is limited by the frequency of its rarest allele, so that SNPswith low frequency alleles are particularly useful as markers of lowfrequency haplotypes. As particular mutations or polymorphismsassociated with certain clinical features, such as adverse or abnormalevents, are likely to be of low frequency within the population, lowfrequency SNPs may be particularly useful in identifying these mutations(for examples see: Linkage disequilibrium at the cystathionine betasynthase (CBS) locus and the association between genetic variation atthe CBS locus and plasma levels of homocysteine. Ann Hum Genet (1998)62:481-90, De Stefano V, Dekou V, Nicaud V, Chasse J F, London J,Stansbie D, Humphries S E, and Gudnason V; and Variation at the vonwillebrand factor (vWF) gene locus is associated with plasma vWF:Aglevels: identification of three novel single nucleotide polymorphisms inthe vWF gene promoter. Blood (1999) 93:4277-83, Keightley A M, Lam Y M,Brady J N, Cameron C L, Lillicrap D).

[0075] According to another aspect of the present invention there isprovided a polynucleotide sequence comprising any one of the intronsequences of HMG-CoA reductase defined in any one of SEQ ID NOS: 18-41herein, an allelic variant thereof, a complementary strand thereof or afragment thereof. A fragment is at least 17 bases, more preferably atleast 20 bases, more preferably at least 30 bases. Preferably theallelic variant is one of the SNPs described herein.

[0076] According to another aspect of the invention there is provided apolynucleotide sequence comprising any one of the intron sequences ofHMG-CoA reductase defined in any one of SEQ ID NOS: 18-41 and 54 or acomplementary strand thereof or a sequence at least 90% homologousthereto.

[0077] The degree of homology may be any of the following: at least 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% homology. Homology is determined asfollows. “Homology” is a measure of the identity of nucleotide sequencesor amino acid sequences. In order to characterize the homology, subjectsequences are aligned so that the highest order homology (match) isobtained. “Identity” per se has an art-recognized meaning and can becalculated using published techniques. Computer program methods todetermine identity between two sequences, for example, include DNAStarsoftware (DNAStar Inc., Madison, Wis.); the GCG program package(Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387); BLASTP,BLASTN, FASTA (Atschul, S. F. et al., J Molec Biol (1990) 215:403).Homology (identity) as defined herein is determined conventionally usingthe well known computer program, BESTFIT (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for example, about 80% homologous to a referencesequence, according to the present invention, the parameters are setsuch that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence or amino acid sequence and thatgaps in homology of up to about 20% of the total number of nucleotidesin the reference sequence are allowed. Eighty percent of homology istherefore determined, for example, using the BESTFIT program withparameters set such that the percentage of identity is calculated overthe fill length of the reference sequence and gaps of up to 20% of thetotal number of amino acids in the reference sequence are allowed, andwherein up to 20% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 20% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. When comparing twosequences, the reference sequence is generally the shorter of the twosequences. This means that for example, if a sequence of 50 nucleotidesin length with precise complementarity to a 50 nucleotide region withina 100 nucleotide polypeptide is compared there is 100% identity/homologyas opposed to only 50% identity/homology. Percent homologies arelikewise determined, for example, to identify preferred species, withinthe scope of the claims appended hereto, which reside within the rangeof about 80 percent to 100 percent homology.

[0078] According to another aspect of the invention there is provided apolynucleotide sequence comprising any one of the intron sequences ofHMG-CoA reductase defined in any one of SEQ ID NOS: 18-41 and 54 or acomplementary strand thereof or a sequence that hybridises thereto understringent conditions. As used herein, stringent conditions are thoseconditions which enable sequences that possess at least 80%, preferablyat least 90% and more preferably at least 95% sequence homology tohybridise together. Thus, nucleic acids which can hybridise to thenucleic acid of SEQ ID No. 18-41 or 54, or the complementary strandthereof, include nucleic acids which have at least 80%, preferably atleast 90%, more preferably at least 95%, still more preferably at least98% sequence homology and most preferably 100% homology. An example of asuitable hybridisation solution when a nucleic acid is immobilised on anylon membrane and the probe nucleic acid is greater than 500 bases orbase pairs is: 6×SSC (saline sodium citrate), 0.5% SDS (sodium dodecylsulphate), 100 mg/ml denatured, sonicated salmon sperm DNA. Thehybridisation being performed at 68° C. for at least 1 hour and thefilters then washed at 68° C. in 1×SSC, or for higher stringency,0.1×SSC/0.1% SDS. An example of a suitable hybridisation solution when anucleic acid is immobilised on a nylon membrane and the probe is anoligonucleotide of between 12 and 50 bases is: 3M trimethylammoniumchloride (TMACl), 0.01M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6),0.5% SDS, 100 mg/ml denatured, sonicated salmon sperm DNA and 0.1 driedskimmed milk. The optimal hybridisation temperature (Tm) is usuallychosen to be 5° C. below the Ti of the hybrid chain. Ti is theirreversible melting temperature of the hybrid formed between the probeand its target. If there are any mismatches between the probe and thetarget, the Tm will be lower. As a general guide, the recommendedhybridisation temperature for 17-mers in 3M TMACl is 48-50° C.; for19-mers, it is 55-57° C.; and for 20-mers, it is 58-66° C.

[0079] Novel sequence disclosed herein, may be used in anotherembodiment of the invention to regulate expression of the gene in cellsby the use of anti-sense constructs. To enable methods ofdown-regulating expression of the gene of the present invention inmammalian cells, an example antisense expression construct can bereadily constructed for instance using the pREP10 vector (InvitrogenCorporation). Transcripts are expected to inhibit translation of thegene in cells transfected with this type construct. Antisensetranscripts are effective for inhibiting translation of the native genetranscript, and capable of inducing the effects (e.g., regulation oftissue physiology) herein described. Oligonucleotides which arecomplementary to and hybridizable with any portion of novel gene mRNAdisclosed herein are contemplated for therapeutic use. Suitableantisense targets include novel intron/exon junctions disclosed herein.U.S. Pat. No. 5,639,595, Identification of Novel Drugs and Reagents,issued Jun. 17, 1997, wherein methods of identifying oligonucleotidesequences that display in vivo activity are thoroughly described, isherein incorporated by reference. Expression vectors containing randomoligonucleotide sequences derived from previously known polynucleotidesare transformed into cells. The cells are then assayed for a phenotyperesulting from the desired activity of the oligonucleotide. Once cellswith the desired phenotype have been identified, the sequence of theoligonucleotide having the desired activity can be identified.Identification may be accomplished by recovering the vector or bypolymerase chain reaction (PCR) amplification and sequencing the regioncontaining the inserted nucleic acid material.

[0080] Antisense nucleotide molecules can be synthesized for antisensetherapy. These antisense molecules may be DNA, stable derivatives of DNAsuch as phosphorothioates or methylphosphonates, RNA, stable derivativesof RNA such as 2′-O-alkylRNA, or other oligonucleotide mimetics. U.S.Pat. No. 5,652,355, Hybrid Oligonucleotide Phosphorothioates, issuedJul. 29, 1997, and U.S. Pat. No. 5,652,356, Inverted Chimeric and HybridOligonucleotides, issued Jul. 29, 1997, which describe the synthesis andeffect of physiologically-stable antisense molecules, are incorporatedby reference. Antisense molecules may be introduced into cells bymicroinjection, liposome encapsulation or by expression from vectorsharboring the antisense sequence.

[0081] According to another aspect of the present invention there isprovided a computer readable medium comprising at least one novelsequence of the invention stored on the medium. The computer readablemedium may be used, for example, in homology searching, mapping,haplotyping, genotyping or pharmacogenetic analysis.

[0082] According to another aspect of the present invention there isprovided a method of treating a human in need of treatment with aHMG-CoA reductase inhibitor drug in which the method comprises:

[0083] i) diagnosis of a single nucleotide polymorphism in HMG-CoAreductase gene in the human, which diagnosis preferably comprisesdetermining the sequence of the nucleic acid at one or more of thefollowing positions:

[0084] position 1962 in the coding sequence of the HMG-CoA reductasegene as defined by the position in SEQ ID NO: 44, and/or

[0085] positions 46 or 267 in the promoter sequence of the HMG-CoAreductase gene as defined by the positions in SEQ ID NO: 45; and/or

[0086] position 129 in intron 2 as defined by the position in SEQ IDNO:20,

[0087] position 550 in intron 5 as defined by the position in SEQ ID NO:24,

[0088] position 37 in intron 15 as defined by the position in SEQ IDNO:37, or

[0089] position 345 in intron 18 as defined by the position in SEQ IDNO:40 of the HMG-CoA reductase gene.

[0090] and determining the status of the human by reference topolymorphism in the HMG-CoA reductase gene; and

[0091] ii) administering an effective amount of a HMG-CoA reductaseinhibitor.

[0092] Preferably determination of the status of the human is clinicallyuseful. Examples of clinical usefulness include deciding whichantagonist drug or drugs to administer and/or in deciding on theeffective amount of the drug or drugs. Statins already approved for usein humans include atorvastatin, cerivastatin, fluvastatin, pravastatinand simvastatin. The reader is referred to the following references forfurther information on HMG-CoA reductase inhibitors: Drugs and TherapyPerspectives (May 12, 1997), 9: 1-6; Chong (1997) Pharmacotherapy17:1157-1177; Kellick (1997) Formulary 32: 352; Kathawala (1991)Medicinal Research Reviews, 11: 121-146; Jahng (1995) Drugs of theFuture 20: 387-404, and Current Opinion in Lipidology, (1997), 8,362-368. Another statin drug of note is compound 3a (S-4522) in Watanabe(1997) Bioorganic and Medicinal Chemistry 5: 437-444.

[0093] According to another aspect of the present invention there isprovided use of a HMG-CoA reductase antagonist drug in preparation of amedicament for treating a HMG-CoA reductase mediated disease in a humandiagnosed as having a single nucleotide polymorphism therein, preferablyat one or more of the positions defined herein.

[0094] According to another aspect of the present invention there isprovided a pharmaceutical pack comprising HMG-CoA reductase antagonistdrug and instructions for administration of the drug to humansdiagnostically tested for a single nucleotide polymorphism therein,preferably at one or more of the positions defined herein.

[0095] According to another aspect of the present invention there isprovided an allelic variant of human HMG-CoA reductase polypeptidehaving a valine at position 638 or a fragment thereof comprising atleast 10 amino acids provided that the fragment comprises the allelicvariant at position 638.

[0096] Fragments of polypeptide are at least 10 amino acids, morepreferably at least 15 amino acids, more preferably at least 20 aminoacids.

[0097] According to another aspect of the present invention there isprovided an antibody specific for an allelic variant of human HMG-CoAreductase polypeptide having a valine at position 638 or a fragmentthereof comprising at least 10 amino acids provided that the fragmentcomprises the valine at position 638.

[0098] Antibodies can be prepared using any suitable method. Forexample, purified polypeptide may be utilized to prepare specificantibodies. The term “antibodies” is meant to include polycionalantibodies, monoclonal antibodies, and the various types of antibodyconstructs such as for example F(ab′)₂, Fab and single chain Fv.Antibodies are defined to be specifically binding if they bind the 1638Vvariant of HMG-CoA reductase with a K_(a) of greater than or equal toabout 10⁷ M⁻¹. Affinity of binding can be determined using conventionaltechniques, for example those described by Scatchard et al., Ann. N.Y.Acad. Sci., 51:660 (1949).

[0099] Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, antigen is administered to the host animal typically throughparenteral injection. The immunogenicity of antigen may be enhancedthrough the use of an adjuvant, for example, Freund's complete orincomplete adjuvant. Following booster immunizations, small samples ofserum are collected and tested for reactivity to antigen. Examples ofvarious assays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

[0100] Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), (1980).

[0101] The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7: 394 (1989).

[0102] Once isolated and purified, the antibodies may be used to detectthe presence of antigen in a sample using established assay protocols,see for example “A Practical Guide to ELISA” by D. M. Kemeny, PergamonPress, Oxford, England.

[0103] According to another aspect of the invention there is provided adiagnostic kit comprising an antibody of the invention.

[0104] The invention will now be illustrated but not limited byreference to the following Examples. All temperatures are in degreesCelsius.

[0105] In the Examples below, unless otherwise stated, the followingmethodology and materials have been applied.

[0106] AMPLITAQ™ available from Perkin-Elmer Cetus, is used as thesource of thermostable DNA polymerase.

[0107] General molecular biology procedures can be followed from any ofthe methods described in “Molecular Cloning—A Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989).

[0108] Electropherograms were obtained in a standard manner: data wascollected by ABI377 data collection software and the wave form generatedby ABI Prism sequencing analysis (2.1.2).

EXAMPLE 1

[0109] Identification of Polymorphisms

[0110] 1. Methods

[0111] DNA Preparation

[0112] DNA was prepared from frozen blood samples collected in EDTAfollowing protocol I (Molecular Cloning: A Laboratory Manual, p392,Sambrook, Fritsch and Maniatis, 2^(nd) Edition, Cold Spring HarborPress, 1989) with the following modifications. The thawed blood wasdiluted in an equal volume of standard saline citrate instead ofphosphate buffered saline to remove lysed red blood cells. Samples wereextracted with phenol, then phenol/chloroform and then chloroform ratherthan with three phenol extractions. The DNA was dissolved in deionisedwater.

[0113] Template Preparation

[0114] Templates were prepared by PCR using the oligonucleotide primersand annealing temperatures set out below. The extension temperature was72° and denaturation temperature 94°. Generally 50 ng of genomic DNA wasused in each reaction and subjected to 35 cycles of PCR. Where describedbelow, the primary fragment was diluted 1/100 and two microlitres wereused as template for amplification of secondary fragments. PCR wasperformed in two stages (primary fragment then secondary fragment) toensure specific amplification of the desired target sequence.

[0115] Single Nucleotide Polymorphism at Position 1962 of SEQ ID NO: 44

[0116] This polymorphism was detected by amplification of a primaryfragment from genomic DNA, followed by amplification of a secondaryfragment, followed by dye primer sequencing with M13F primer:

[0117] Primary Fragment

[0118] Forward Oligo, SEQ ID NO: 1

[0119] Reverse Oligo, SEQ ID NO: 2

[0120] Annealing Temp 68°

[0121] Time 1 min

[0122] Secondary Fragment

[0123] Forward Oligo, SEQ ID NO: 3

[0124] Reverse Oligo, SEQ ID NO: 4

[0125] Annealing Temp 69°

[0126] Time 1 min

[0127] Single Nucleotide Polymorphisms at Positions 46 and 267 of SEQ IDNO: 45

[0128] These polymorphisms were detected by amplification of a primaryfragment from genomic DNA, followed by dye terminator sequencing usingthe same oligos.

[0129] Forward Oligo SEQ ID NO: 5

[0130] Reverse Oligo SEQ ID NO: 6

[0131] Annealing Temp 64°

[0132] Time 2 min

[0133] Single Nucleotide Polymorphisms at Position 129 of HMG CoAReductase Intron 2 Sequence (SEQ ID NO: 20)

[0134] This polymorphism was detected by amplification of a primaryfragment from genomic DNA, followed by dye terminator sequencing.

[0135] Primary Fragment

[0136] Forward Oligo SEQ ID NO: 7

[0137] Reverse Oligo SEQ ID NO: 8

[0138] Annealing Temp 53°

[0139] Time 1 min

[0140] Dye terminator sequencing oligo SEQ ID NO: 9

[0141] Single Nucleotide Polymorphisms at Position 550 of HMG CoAReductase Intron 5 Sequence SEQ ID NO: 24 (T to A).

[0142] This polymorphism was detected by amplification of a primaryfragment from genomic DNA, followed by dye primer sequencing with M13Fprimer:

[0143] Primary Fragment

[0144] Forward Oligo SEQ ID NO: 42

[0145] Reverse Oligo SEQ ID NO: 43

[0146] Annealing Temp 69°

[0147] Time 1 min

[0148] Single Nucleotide Polymorphisms at Position 37 of HMG CoAReductase Intron 15 Sequence SEQ ID NO: 37 (A to G).

[0149] This polymorphism was detected by amplification of a primaryfragment from genomic DNA, followed by amplification of a secondaryfragment, followed by dye primer sequencing with M13F primer:

[0150] Primary Fragment

[0151] Forward Oligo SEQ ID NO: 10

[0152] Reverse Oligo SEQ ID NO: 11

[0153] Annealing Temp 68°

[0154] Time 1 min

[0155] Secondary Fragment

[0156] Forward Oligo SEQ ID NO: 12

[0157] Reverse Oligo SEQ ID NO: 13

[0158] Annealing Temp 69°

[0159] Time 1 min

[0160] Single Nucleotide Polymorphisms at Position 345 of HMG CoAReductase Intron 18 Sequence

[0161] This polymorphism was detected by amplification of a primaryfragment from genomic DNA, followed by dye terminator sequencing.

[0162] Primary Fragment

[0163] Forward Oligo SEQ ID NO: 14

[0164] Reverse Oligo SEQ ID NO: 15

[0165] Annealing Temp 58°

[0166] Time 1 min

[0167] Dye Terminator Sequencing Oligo SEQ ID NO: 16

[0168] Dye Primer Sequencing

[0169] Dye-primer sequencing using M13 forward and reverse primers wasas described in the ABI protocol P/N 402114 for the ABI Prism™ dyeprimer cycle sequencing core kit with “AmpliTaq FS”% DNA polymerase,modified in that the annealing temperature was 45° and DMSO was added tothe cycle sequencing mix to a final concentration of 5%.

[0170] The extension reactions for each base were pooled, ethanol/sodiumacetate precipitated, washed and resuspended in formamide loadingbuffer.

[0171] 4.25% Acrylamide gels were run on an automated sequencer (ABI377, Applied Biosystems).

[0172] Dye Terminator Sequencing

[0173] Dye-terminator sequencing was as described in the ABI protocolP/N 4303150 for the ABI Prism™ Big Dye terminator cycle sequencing corekit with “AmpliTaq FS”™ DNA polymerase.

[0174] The extension reactions were ethanol/sodium acetate precipitated,washed and resuspended in formamide loading buffer.

[0175] 4.25% Acrylamide gels were run on an automated sequencer (ABI377, Applied Biosystems).

[0176] 2. Results

[0177] Exon-Intron Organisation of the Human HMG-CoA Reductase Gene

[0178] Exon sequences are in capital letters: intron sequences (whereshown) are in lowercase letters. The number shown immediately below theDNA sequence denotes the nucleotide position from SEQ ID NO: 44 at whichthe intron interrupts the HMG CoA reductase mRNA. The 5′ boundary andsequence of intron 1 are as described by K. L. Luskey, Mol. Cell. Biol.7:1881-1893 (1987), Medline ref. No.87257890. Sequence of Exon-IntronJunctions Intron no. 5′ Boundary               3′ Boundary Intron size(Kb) 1.    GAT CTG GAG gtgagg(SEQ ID NO: 17)..ATG TTG TCA 4.5 approx                                      51 2.    TTT GAG GAG ..........GAT GTT TTG 1.2 approx            215            216 3.    ATA TTT TGG.............. GTA TTG CTG 0.28            327                328 4.   AGG CTT GAA ............ TGA AGC TTT 1.222           415              416 5.    AAC TCA CAG ............ GAT GAAGTA 1.7 approx            500              501 6.    CCA TGT CAG .....GGG TAC GTC 2 approx            606       607 7.    GTA TTA GAG............. CTT TCT CGG 0.11            713               714 8.   ATG ATT ATG ...... TCT CTA GGC 0.414            830        831 9.   TCT CTC TAA ........... AAT GAT CAG 0.12           991             992 10.    AAA GAA AAG ...... TTG AGG TTA0.108            1239       1240 11.    AAT GCA GAG ...........AAA GGTGCA 4 approx            1418           1419 12.    TAC TCC TTG........... GTG ATG GGA 0.358            1613           1614 13.    GCAATA GGT ............. CTT GGT GGA 0.15           1772              177314.    CAC TAG CAG .......... ATT TGC ACG 1.5 approx          1930            1931 15.    ATT TCA AAG ...... GGT ACA GAG 2approx            2036       2037 16.    GTC AGA GAA .... GTA TTA AAG0.343            2207     2208 17.    TGT GGA CAG ........ GAT GCA GCA0.088            2348          2349 18.    TGT TTG CAG ...... ATG CTAGGT 0.428           2507        2508 19.    TCA CCA CAG ...... GTC GAACAT 0.149          2662         2663

[0179] Polymorphisms SEQ ID NO: 44 Nucleotide 1962 A/G Ile/Val (638)ATA/GTA ATA 95.5% GTA 4.5%

[0180] The allele frequencies were based on analysis of 22 individuals.A was the published base. This change in amino acid sequence is withinthe catalytic domain of the polypeptide and may therefore be ofparticular interest. SEQ ID NO: 45 Nucleotide 46 C/G Allele Frequency C95.8% G 4.2% C was the published base. Nucleotide 267 T/C AlleleFrequency T 95.8% C 4.2%

[0181] T was the published base. These changes in the promoter mayaffect transcript levels. The allele frequencies were based on analysisof 24 individuals.

[0182] HMG CoA Reductase Intron 2 Sequence Nucleotide 129 of SEQ ID NO:20 Insertion of AA Allele Frequency CT 95% CAAT 5%

[0183] Allele frequencies determined in a panel of 20 individuals

[0184] HMG CoA Reductase Intron 5 Sequence Nucleotide 570 of T/A SEQ IDNO: 24 Allele Frequency T 72.7% A 27.3%

[0185] The allele frequencies were based on analysis of 22 individuals.

[0186] HMG CoA Reductase Intron 15 Sequence Nucleotide 37 of A/G SEQ IDNO: 37 Allele Frequency A 97.7% G 2.3%

[0187] The allele frequencies were based on analysis of 22 individuals.

[0188] HMG CoA Reductase Intron 18 Sequence Nucleotide 345 of SEQ ID NO:40 T/C Allele Frequency C 61.7% T 28.3%

[0189] The allele frequencies were based on analysis of 23 individuals.

[0190] Summary of Polymorphisms SNP Ref Position Change Exon 15 SEQ IDNO: 44 1962, 638 A → G, Ile → Val promoter SEQ ID NO: 45 46 C → Gpromoter SEQ ID NO: 45 267 T → C Intron 2 SEQ ID NO: 20 129 CT → CAATIntron 5 SEQ ID NO: 24 550 T → A Intron 15 SEQ ID NO: 37 37 A → G Intron18 SEQ ID NO: 40 345 T → C

[0191] Intron Sequence

[0192] Intron 1 Sequence (Last 634 bp)

[0193] SEQ ID NO: 18

[0194] Intron 2 Sequence

[0195] First 506 bp, SEQ ID NO: 19

[0196] Last 230 bp, SEQ ID NO: 20

[0197] Intron 3 Sequence (280 bp)

[0198] SEQ ID NO: 21

[0199] Intron 4 Sequence (1,222 bp)

[0200] SEQ ID NO: 22

[0201] Intron 5 Sequence (First 850 bp and Last 730 bp)

[0202] SEQ ID NO: 23

[0203] SEQ ID NO: 24

[0204] Intron 6 Sequence (First 492 bp and Last 715 bp)

[0205] SEQ ID NO: 25

[0206] SEQ ID NO: 26

[0207] Intron 7 Sequence (109 bp

[0208] SEQ ID NO: 27

[0209] Intron 8 Sequence (414 bp)

[0210] SEQ ID NO: 28

[0211] Intron 9 Sequence (118 bp)

[0212] SEQ ID NO: 29

[0213] Intron 10 Sequence (108 bp)

[0214] SEQ ID NO: 30

[0215] Intron 11 Sequence (First 728 bp and Last 291 bp¹)

[0216] SEQ ID NO: 31

[0217] SEQ ID NO: 54

[0218] Intron 12 Sequence (358 bp)

[0219] SEQ ID NO: 33

[0220] Intron 13 Sequence (150 bp)

[0221] SEQ ID NO: 34

[0222] Intron 14 Sequence (First 247 bp and Last 594 bp)

[0223] SEQ ID NO: 35

[0224] SEQ ID NO: 36

[0225] Intron 15 Sequence (First 357 bp)

[0226] SEQ ID NO: 37

[0227] Intron 16 Sequence (342 bp)

[0228] SEQ ID NO: 38

[0229] Intron 17 Sequence (87 bp)

[0230] SEQ ID NO: 39

[0231] Intron 18 (427 bp)

[0232] SEQ ID NO: 40

[0233] Intron 19 Sequence (148 bp)

[0234] SEQ ID NO: 41

EXAMPLE 2

[0235] Single Nucleotide Polymorphism at Position 915 of HMG CoAReductase Intron 4 Sequence SEQ ID No: 22 (Deletion of T)

[0236] This polymorphism was detected by amplification of a primaryfragment of genomic DNA, followed by a secondary fragment, followed bydye terminator sequencing.

[0237] a) Primary Fragment

[0238] Forward oligo SEQ ID No: 49, Reverse oligo SEQ ID No: 47

[0239] Annealing temperature 55° C., Time 1 min

[0240] b) Secondary Fragment

[0241] Forward oligo SEQ ID No. 48, Reverse oligo SEQ ID No. 46

[0242] Annealing temperature 55° C., Time 1 min

[0243] Dye terminator sequencing oligo; SEQ ID No: 50

Example 3

[0244] ARMS™ Diagnostic Assay To Detect Exon 15 Polymorphism

[0245] ARMS™ assay technology is described in Chapter 11 of the textbookPCR by C R Newton & A Graham, 2^(nd) Edition, BIOS Scientific PublishersLtd, Oxford, UK. Below are the primer sequences needed to carry out adiagnostic ARMS™ assay on the exon 15 polymorphism, in order to detectwhich allele is present.

[0246] The following primers amplify a 198 base pair PCR product only ifthe A allele is present:

[0247] Constant primer (forward): SEQ ID NO: 51

[0248] A allele specific primer (reverse): SEQ ID NO: 52

[0249] Annealing temp. 68° C., Time 45 secs

[0250] The following primers amplify a 198 base pair PCR product only ifthe G allele is present:

[0251] Constant primer (forward): SEQ ID NO: 51

[0252] G allele specific primer (reverse): SEQ ID NO: 53

[0253] Annealing temp. 68° C., Time 45 secs

[0254] Sequence Listing Free Text

[0255] For SEQ ID NO: 46-49 & 51-53:

[0256] <223> Description of Artificial Sequence:PCR primer

[0257] For SEQ ID NO: 50:

[0258] <223> Description of Artificial Sequence:dye terminatorsequencing oligo

1 54 1 30 DNA Homo sapiens 1 gtggatgttg cagtgagcca agatcaagcc 30 2 33DNA Homo sapiens 2 cgtcctaagt aaacccagga tatgtgtaat gcc 33 3 24 DNA Homosapiens 3 ctccagcctg ggccacagag tgag 24 4 51 DNA Homo sapiens 4tgtaaaacga cggccagtcg tcctaagtaa acccaggata tgtgtaatgc c 51 5 40 DNAHomo sapiens 5 accaggaaac agctatgacc ctatcgcctc cgcctagcag 40 6 38 DNAHomo sapiens 6 actgtaaaac gacggccagt ctcccaccca tctcgccc 38 7 24 DNAHomo sapiens 7 attggacttg tagtgtgctt acat 24 8 24 DNA Homo sapiens 8tccaaataca tgatttgaat gaac 24 9 19 DNA Homo sapiens 9 catgatttgaatgaacagg 19 10 30 DNA Homo sapiens 10 gtggatgttg cagtgagcca agatcaagcc30 11 33 DNA Homo sapiens 11 cgtcctaagt aaacccagga tatgtgtaat gcc 33 1224 DNA Homo sapiens 12 ctccagcctg ggccacagag tgag 24 13 51 DNA Homosapiens 13 tgtaaaacga cggccagtcg tcctaagtaa acccaggata tgtgtaatgc c 5114 25 DNA Homo sapiens 14 tgctaggtgt tcaaggagca tgcaa 25 15 25 DNA Homosapiens 15 ttcaggctgt cttcttggtg caagc 25 16 25 DNA Homo sapiens 16gggaccgtaa tggctgggga attgt 25 17 15 DNA Homo sapiens 17 gatctggaggtgagg 15 18 635 DNA Homo sapiens UNSURE 16 any 18 tttttttctg ttgcancaatgtaggaaggt ttattaacca tccttttgct agtgacatta 60 tgccatatgt tctatggaatgaaaaagtac aagaggccct gcccttgaga tcttatcaac 120 taacatgatt tatagcaggncctcaataag tggattcttg ggtgtttacc ttttgtgtaa 180 tcagaatgta gatgatgaagaagatactta acatgcattt tatatctagg taattagaaa 240 atgtgaatag ctgtttctcacttgtgtttt ctgcttgatt gctcttctac ttgcaaggct 300 taggtaataa ggtcgagatacttatctggt ttgatcttaa atgtttgaat tcatataatt 360 tttaagaaat ggctgctttaaagttggttg ccagtaagta ataaaggatt tattgtttga 420 gtgaagaaga aataacatagttctcttaat tttataatta ttttccagaa ttataaggaa 480 cagtatcaaa tagtcatatgtatgggacac tgtgcataca aagcagggtt tatagcacac 540 ttttccttaa aatcttttcctaaaaataca atgagctgta tactaagtgt tcacccttga 600 tattccttcc aggatccaaggattctgtag ctaca 635 19 506 DNA Homo sapiens 19 gttagtgaag ttaatttgatactgactaaa gtaaattaca ttttcaattt ttgaagagcc 60 cttaagcccc tatagggagcacataatttt taaaagttag agtaaaatat ttattttagt 120 attttggaac ttacctcaaatttctgctta catggaatgc actggaaatg ctcttatttt 180 gctttgtctt tacaaatgaatgtaattgac tttatttgag aaatacatct tttataagtg 240 actaatagtc aaaaatgattgtgggccggg tatagtgact catgcctgta atcccagcac 300 tttgggaggc caaagcaggagaattgtttg agcctaggag ttcaagacca gcctgggcaa 360 cctggacaac atagtaagacccaagtcttt aaaaaaaaat taaaggccgg gcaccggtgg 420 ctcatgtctg taatcccagcactttgggag ggcaaggcgg gtggatcacg aagtcgggag 480 aatcgagacc attctggctaacatgg 506 20 230 DNA Homo sapiens UNSURE 26 any 20 aaaaaaaaaaaattgtggtg atgtantggc ttnccacagt ggtttgatta aaagttggat 60 ttaatttttgatttgtaggt ttgatatttt tattggcttg tagtgtgctt acatttatgt 120 tctcatgactatataaatga attacacatg caaaataaaa attcttagtt ttgattactt 180 attttaaaagtcaaagctaa tggaatttcc ttttctttct ctcctattag 230 21 280 DNA Homo sapiens21 ctaaaatgac aaaagttcaa tactaaaaaa actttatcct ttactacaca aataataaca 60gtgtcacaca gcattctgag ataatactag tttactccaa attattaagg tctcaaattt 120cagaatgtct aattgccaat aataaggaaa ctattcaatg catgcagcac actttcagca 180atacacatat ttccaaatac atgatttgaa tgaacaggtt tatttccaca gcaaatcaaa 240aatctgatga caccagaagt taaatatgta aaactattac 280 22 1222 DNA Homo sapiensUNSURE 604 any 22 gtaagtattt aaaacctaaa tatactttct gtcaaaatac attttaaaaaacttttcttc 60 cccatgctgt aaaggtacat tttcaaaagt taagaaaata aggggaaatttttttgtata 120 attttactat tagctaattt taataactat taacattttg gcatatatccttttctactg 180 tttttatact taaagaaaat atctgatatc atatatattg ttttataatttctttatgct 240 taataatagt ttatcaacat ctttccatgt cccttttttt tttttttgagatggagtttc 300 gctcttgtca cccaggctgg agtgtaatgg cacgatcctg gctcactgcaacctccactt 360 cccgggttca agcagttctc ctgcctcagc ctcctgagta gctgggattcaggcacctgc 420 caccatgccc atttaatttg tgtatttttg gtagagactg tgtttcgcatgttggcaggc 480 tggggcaaac tcctgcctca agtgatccgc ctgccttggc ctccaaagtctgggattata 540 ggcgtgagcc ctggcccggc ctcatgtcct taatgtaaac taaatggttggaatggctat 600 atgnatctct tctgctaaac gcttggagnt attaatcatc taatgtggactgttaggtat 660 gtaatttttt ttattaatac caccactgtg atgaatgtct ttgaacgaaatttttgttca 720 tatttgtaat cattttctta agatacattc ctagaagtga gacagtggttttgctttttt 780 tagagctttg cttttttttt tttaagagct ttttagtgct actattgccaatttagttta 840 cagaaagttt gtttagttta tccttccgca ggtagtgatc aatgaaaatttttatatatt 900 ccactttttt tccctaatgg taatccaagg agatattttt tactaaggatgatactttga 960 tacaaaatta tcaaaaggtg tttaaatgta aatatactta cattttaacattaaaaatat 1020 ttttaacaaa tattttgagc aactactatg tttagctttg aggatgccaaagaaatatag 1080 ggtatacttt tttgtctgca gaaaggtaca catactacag gatcatacagtatggagggg 1140 gaaaggtttt gttctaaaaa gaattttttt aaaatcatac tttttcccgttaaatttatc 1200 tgatcatttt gttcttttcc ag 1222 23 849 DNA Homo sapiensUNSURE 1 any 23 nnannnttat tatccgatgg antgngaatn gnnttccttt nttcagncnacattaatagg 60 aaggattaat ngcgtttctt catagcacaa gatttaagaa ttgcccaaagttttaagtnt 120 aattctcaag cccaagactg gtctccataa gtgccccagg aacagtcccctgtatctaac 180 aactcaatta tgattctgta gctactggaa tttggaattn cccccatttttctttttgaa 240 agttttcaga acttntggtn ataataattt ttgggtnaat aagagtattttcctagtgaa 300 acacatcaga gagcagaaca agatctaatg gaagagaaac ccagggtagattgattgatt 360 gattgatttg agatgtcgtc tcacnctnct acccngngct gnnttgctcnnggtnagatc 420 ttnggtcacc caccnccctt ntcactcntg gctncgcacg ggntccttctgccntcnccc 480 acctnnncag cnnncgattc cnccntggtn cccctnccnt ctcgcnnctnaatcttntcc 540 tgttcncatg ctctncncnn tattctttng ccantgnttg gctcaagactcgccccttca 600 tnttctttgc accntcaann cgnacccncc cgcnctcccc cttccctagtgcgcgngnat 660 gttcngcttn ganccnctnc tncctgngcc atntttnntc ttanaccccnnaaatncacn 720 cttggccctt tntnntccta ttnttacnct tcnntctttn acgtgncactntntancttt 780 tnacnnaccc tnancctctt tnccacntnt cctnttnnct atnnctctgcctcttnannt 840 nccccacct 849 24 730 DNA Homo sapiens UNSURE 8 any 24cgaaaaangc cccattcncc cnatattggn aagaaggggg atcnttgaca tatacaagga 60ataggtgggt ttatgaatcn aatangtctc ataaatttcn aactaagctt ctgtcaggta 120ggtaaaatat agaangtgna ggatttaatt aggggattaa atgccagagt aatnccctnc 180ctcaaaggaa tantcctacc aaatatcnna ttcagggaaa ggaatcaggg cnctatatgt 240tcttttttaa aattgggcng ggcccagggc tcncacctga attccagcac ttcggnagnc 300cgaggtgggc agatcncctg aggcaggagc tccagaccag cctggccaac ctggtgaaac 360ccagtctcta ctaaaaatac aaaaattagc tgggcatggt ggcgggtgcc tgtaatccca 420gctactcggg aggctgaggc aggagaattg cttgaaccca ggaggcagag gtngcagtga 480accaagatca caccattgca cattgcactc cagcctggga aacaaagtga gactacatct 540caaaaaaaaa tttttttaaa tcctttatat tacaatcata ctttgtatct tgaatacctg 600ttagttttat catattgtat attttactct ttgaatagta atttgatatt aatataagcc 660ataggatgct ctaacattta aaaaagttgt tctgtccctt gccttcattg atatgtttga 720tctgttttag 730 25 491 DNA Homo sapiens UNSURE 88 any 25 gtttgtaagcaatttttgcc atattttaaa ataggtatgt cgctaaagag gaaaaagaac 60 atcttggatttgtattattt attgttanat tctgactttt aaattactct taaaattttt 120 tattatattagtgtgtgggt atatctggcc tgtttgcttt ggtggaaact tagcagcagg 180 ttactgatttatttttcact cctgccanct actttgtgng catnactntg tgatattttt 240 attcattngatntctttngt tnggtttttt anccaacatt gcattccaag gttggtctgg 300 aaaacactttccagcccctg ctgctactta agactcaatt ggngacttgg gtctttgtgt 360 nttaattntcatgtagggac gaaaagagtc aggaattggg accagatctg gagtaaggat 420 tcacctnataaggnggattg ggaantattt aaatccggat aagtaagccg gaccatcttc 480 agcaagtacc c491 26 715 DNA Homo sapiens 26 cgttttctat tttgagatcc cccaaatgaatccctttaac aagtgtgaag tacaatatgt 60 ttggttaatt tttagtctta agttgggcattttgtattat atcattttcc aacagatcaa 120 tgaaataacc aaaattataa gaacttcagtaggcatcata gaggttatat tgaaaattag 180 agtttgttgg tacacaaaaa atattattttgaccttatat caggactggc ataactggca 240 ggatattcta cttatataaa aaatccttggttaattggca aattgctttt ctcctaacaa 300 gtgggattag atcaatagtg tcactggggttttgctctgt tagagtagcc tgtcctctcc 360 ttatttaata ataaggcact actgcttgacaaaaaggaat tggaaacaca tatgttttat 420 caattgtgta ttaaacacta ccattctgcctggcattgtg atatggggac atgagagaag 480 gcaagagctt tgctcttgag ggacttagagttctgttgtt attcctgctg tttcctaagg 540 cttggcatca cctctaagtt gctaattctatttccagtaa gtggcaagga gcttaatgta 600 ctaatatttt catgttttgt ccacctgcaggaagacaaat atatccttgt gatatatgca 660 gcataaaaaa taacgtagac tttactagttgtatctttaa tttttctcta accag 715 27 109 DNA Homo sapiens 27 gtaagcccaattcttacata tggcactagt agaagagtaa gatttctgct tacacagttt 60 acctaaacagaatcaatacc ttctaatgtc acactgactt aatttgtag 109 28 414 DNA Homo sapiens28 gtaatgacat ggttttcttc ttcttttagt atcctcagtt ccaatctcat tatttttaag 60atttcttttt ttctacaatt ttggcccatt caatgatatt gcaccccctt cttccttttt 120ttcttaatgt gttcatttct ttgaggctcc tggtctttat tagcccctct ctcctaaaca 180gactttttaa gttcccccac cttatctctc gttgaaagcc tgttctttgg ggtgttttca 240gtagttcagt ggggtcacta ctttagttag ttgcatagca agcttggggc tttttttttt 300tcatggtaag gggaagctat gagagataat gtctggctgt ccagttgcta gggataagaa 360atttaagttc tattgatatg cagaggatac attactttaa aaattttatt tcag 414 29 118DNA Homo sapiens 29 gtaagttaat tgaaatctac tttgtgatat attaatcataacactctatg ctaatgtaag 60 tttagattgt gtcctttaca tttctgaata agattttaatttgctttctt ttatttag 118 30 108 DNA Homo sapiens 30 gtaacttgtt attctcttcgctttcaatcc ttcattgctt tgtcaaaaag tagtctgttt 60 tcaaaattat gtgccgtgttgtgagatttc ttttgatttc ttgaacag 108 31 728 DNA Homo sapiens UNSURE 49 any31 gtgaggatga taacataaac tccaatgtgg catttttcat tacaaaggng cttngnnaag 60gangaaaaat ctagtatctg ctgaacactn cagctaagtt ctgggcacgg tgtancatga 120ctaacagata ctatcttctt tctttatttc acacaacctt gagaggtagg tncaattatc 180tatttttcag atgagaacat tgaggctcca atatgtttaa tttcccaaag nagtccctct 240nggaaatgat naagctgata gnagggtcca agattttctg actccagagt caaaactctt 300tctagtttat tactgcttat catagagatg agtgactact gtatnctcat aggngtgntg 360aggcctagaa agagtttacc acagagacaa gtttcaaaga tagangaaag tttgttttng 420tnttgtttng nngctggata cccatgagga agtttgcttt tctttctgac atttgaacag 480gaccttntgc ctacatgacc atatgaatct acttatgctt tcatgcaaan aatcatggtt 540ccatncatgt ctgcttnaca cgggtgtttc ttttaannca caggntaatt ncgtttaatt 600gggnaaaatg ccattttttg gccagccttt tttgagggtt tcttggccaa antttttttt 660gnatantnnt gatnnataat gattattatc nctngntttg gagacaaaan ntnncttttt 720tcccccag 728 32 30 DNA Homo sapiens 32 caatttcatt ttttttctcc atttctttag30 33 358 DNA Homo sapiens 33 gtatgttatt ttctcgatta agagagatttgctttgtatg tttttaatct tttttcttga 60 ttagtttcat atatgtacat agttttataaaacattttcc ttttaaatca ttttatccta 120 attttttatt ctgcttatga tgtaggtcatagaaattaaa aatatatttc ctgcttttat 180 agtcattact caaagatttt agtattttaaacacttttta aaggtgaatt aaacattttg 240 tttaaaaaga atacatacta aaggattaagtttgaagata gttatactga caagctgaga 300 taaaattttg tgcatttact atatagattttcatttggtg cctgacttta ccttttag 358 34 150 DNA Homo sapiens 34 gtaagttggcatttatatat ttgccagttt aaaaatacat cataagtaag gcaatgagaa 60 gagttttaaggacaattagt gatacctttt gggtcaagca tgagcatttt tgggtaacat 120 gtgcttgcttctctaacata tactgtgtag 150 35 246 DNA Homo sapiens UNSURE 106 any 35gtgtgtgagt ggatttgtat gtacagttat atctatttgt ttattttaga accagtgtca 60ttttctgtga ttaccaaaca taattgttaa catattacct gctaangagc acataacaga 120atatcaactt taaagccatt cattnaaaat gagtaatatt tatgctgggn nggggggaaa 180aaaagaatgt ngatncaaat gaatngctcc ncagaggtaa attagtaaga aaaaaaaaaa 240gggggg 246 36 594 DNA Homo sapiens UNSURE 22 any 36 aaaaaaaaaaaaaaagtgta anctagtaat ttttgattag atgttacttt gcctaggana 60 gaactgttttagaaaaaaag atttttcaaa taggagagaa atattagtat aataagactt 120 ccttcaaataaagaaaatta ataaagtagc ataatcaacn caaatgatan ccatagtata 180 gttcaagcttaacacatttg tttttatgtg aactgtgtaa gtttattaag aaataattgt 240 gactgggcgcagtggctcac gcctgtaatc ccaacacttt ggggaggcca acgcgggcag 300 atcacttgaggccaggagtt cgagaccagc ctggccanca tggcgaaacc ctgtctctac 360 tcaaaatacaaaaattggct gggcatggtg gcccgcgcct gtaatcccag ctactcggga 420 ggctgaggctggagaatttc ttgaacccgg gaggtggatg ttgcagtgag ccaagatcaa 480 gccactgcactccagcctgg gccacagagt gagactccgt ctcaaaaaan aacaaaaaac 540 aaagaaataataataataaa agaataaaac acagtctttg catctttntt atag 594 37 357 DNA Homosapiens UNSURE 6 any 37 gtaagnntng ccagantntn tnaangtcct tttattaantntttnnnctt ttataaaaaa 60 caaatcagcc cttttgttga tggncattcn ttncnttnganngattcant ttanantngg 120 cnttacacat atcctgggtt tacttaggac gggnaacantnttagtntng acatttcaaa 180 actttntcca gtcaananac cncntttgag gctgacctctncaagattng tntttaanan 240 cnccantatn ttttcngcct tnggnaggcc nnggcaagaagnttgnttgg ggtntgaagn 300 tnaanaccag ccngggcnac acananagat gctntntctanaaacaataa aaaaaaa 357 38 342 DNA Homo sapiens UNSURE 39 any 38gtgagtgact ggatggataa tttatctttt ttattttgna atctttaatt gtatttaaaa 60atgggggaaa ggagtattaa cattttaaat aaagttaaat atatgggaca gtgttttcca 120tcaaagatga ctgttgtacc ttgcccatct gtctgtgtgn atcatccata ggaacaaact 180ttactgattt tttttaattt tttttatttt ttaatggagg acagggctta aatggggcca 240catctaaact ttgttttctg gaggttcaga aagatagatt tgggtaacat tcccctgaac 300cttctggagg aacatctaaa tgtacacagc tctgttttgt ag 342 39 87 DNA Homosapiens 39 gtgagctctc cagcctccac ttctcttgtg ttacgtcttt ctaagtgaaagaagtatatg 60 gtatattttt tcttttcttg tttccag 87 40 428 DNA Homo sapiens40 gtatgatgta tcaggcatag agtccacaag cctagttctg actctctggg tttctctttc 60tatctgagac tatgtatcac tcacctctat tttaattggt cttttccaaa ctcttttgtc 120atatcagcct aatccattgt gtccaaataa gcatgtttaa gcttatgctt agataagaaa 180gtagatgaag agagcaaatg aatgttcatc tactgagtta agggtactgc cagtcaggct 240gtgaatatta tgttagctat ggtattatgc actgtcaggt gtggctgtca agtcttggaa 300agttagtgct tccagtggag tctagttcta ttctgatgcc attatagttg ccctgttttt 360agttgattta gtaagaaatt ggtcatgatt ttaagggtga atcttgttgt gtctctccct 420ggctacag 428 41 148 DNA Homo sapiens 41 gtaagactca aagatatatt taacatgttccccctatact tcaaaaaata tgcagtgtaa 60 aaacttacta ttcatctact gtagttccaagttaaaattc tacactcctg atatttatat 120 attgctactt tgtcattttc taccatag 14842 42 DNA Homo sapiens 42 aacgacggcc agttcaggag ctccagccag ctggcaacct gg42 43 31 DNA Homo sapiens 43 ggcaggagtg aaaaataaat cagtaacctg c 31 442904 DNA Homo sapiens 44 ttcggtggcc tctagtgaga tctggaggat ccaaggattctgtagctaca atgttgtcaa 60 gactttttcg aatgcatggc ctctttgtgg cctcccatccctgggaagtc atagtgggga 120 cagtgacact gaccatctgc atgatgtcca tgaacatgtttactggtaac aataagatct 180 gtggttggaa ttatgaatgt ccaaagtttg aagaggatgttttgagcagt gacattataa 240 ttctgacaat aacacgatgc atagccatcc tgtatatttacttccagttc cagaatttac 300 gtcaacttgg atcaaaatat attttgggta ttgctggccttttcacaatt ttctcaagtt 360 ttgtattcag tacagttgtc attcacttct tagacaaagaattgacaggc ttgaatgaag 420 ctttgccctt tttcctactt ttgattgacc tttccagagcaagcacatta gcaaagtttg 480 ccctcagttc caactcacag gatgaagtaa gggaaaatattgctcgtgga atggcaattt 540 taggtcctac gtttaccctc gatgctcttg ttgaatgtcttgtgattgga gttggtacca 600 tgtcaggggt acgtcagctt gaaattatgt gctgctttggctgcatgtca gttcttgcca 660 actacttcgt gttcatgact ttcttcccag cttgtgtgtccttggtatta gagctttctc 720 gggaaagccg cgagggtcgt ccaatttggc agctcagccattttgcccga gttttagaag 780 aagaagaaaa taagccgaat cctgtaactc agagggtcaagatgattatg tctctaggct 840 tggttcttgt tcatgctcac agtcgctgga tagctgatccttctcctcaa aacagtacag 900 cagatacttc taaggtttca ttaggactgg atgaaaatgtgtccaagaga attgaaccaa 960 gtgtttccct ctggcagttt tatctctcta aaatgatcagcatggatatt gaacaagtta 1020 ttaccctaag tttagctctc cttctggctg tcaagtacatcttctttgaa caaacagaga 1080 cagaatctac actctcatta aaaaacccta tcacatctcctgtagtgaca caaaagaaag 1140 tcccagacaa ttgttgtaga cgtgaaccta tgctggtcagaaataaccag aaatgtgatt 1200 cagtagagga agagacaggg ataaaccgag aaagaaaagttgaggttata aaacccttag 1260 tggctgaaac agatacccca aacagagcta catttgtggttggtaactcc tccttactcg 1320 atacttcatc agtactggtg acacaggaac ctgaaattgaacttcccagg gaacctcggc 1380 ctaatgaaga atgtctacag atacttggga atgcagagaaaggtgcaaaa ttccttagtg 1440 atgctgagat catccagtta gtcaatgcta agcatatcccagcctacaag ttggaaactc 1500 tgatggaaac tcatgagcgt ggtgtatcta ttcgccgacagttactttcc aagaagcttt 1560 cagaaccttc ttctctccag tacctacctt acagggattataattactcc ttggtgatgg 1620 gagcttgttg tgagaatgtt attggatata tgcccatccctgttggagtg gcaggacccc 1680 tttgcttaga tgaaaaagaa tttcaggttc caatggcaacaacagaaggt tgtcttgtgg 1740 ccagcaccaa tagaggctgc agagcaatag gtcttggtggaggtgccagc agccgagtcc 1800 ttgcagatgg gatgactcgt ggcccagttg tgcgtcttccacgtgcttgt gactctgcag 1860 aagtgaaagc ctggctcgaa acatctgaag ggttcgcagtgataaaggag gcatttgaca 1920 gcactagcag atttgcacgt ctacagaaac ttcatacaagtatagctgga cgcaaccttt 1980 atatccgttt ccagtccagg tcaggggatg ccatggggatgaacatgatt tcaaagggta 2040 cagagaaagc actttcaaaa cttcacgagt atttccctgaaatgcagatt ctagccgtta 2100 gtggtaacta ttgtactgac aagaaacctg ctgctataaattggatagag ggaagaggaa 2160 aatctgttgt ttgtgaagct gtcattccag ccaaggttgtcagagaagta ttaaagacta 2220 ccacagaggc tatgattgag gtcaacatta acaagaatttagtgggctct gccatggctg 2280 ggagcatagg aggctacaac gcccatgcag caaacattgtcaccgccatc tacattgcct 2340 gtggacagga tgcagcacag aatgttggta gttcaaactgtattacttta atggaagcaa 2400 gtggtcccac aaatgaagat ttatatatca gctgcaccatgccatctata gagataggaa 2460 cggtgggtgg tgggaccaac ctactacctc agcaagcctgtttgcagatg ctaggtgttc 2520 aaggagcatg caaagataat cctggggaaa atgcccggcagcttgcccga attgtgtgtg 2580 ggaccgtaat ggctggggaa ttgtcactta tggcagcattggcagcagga catcttgtca 2640 aaagtcacat gattcacaac aggtcgaaga tcaatttacaagacctccaa ggagcttgca 2700 ccaagaagac agcctgaata gcccgacagt tctgaactggaacatgggca ttgggttcta 2760 aaggactaac ataaaatctg tgaattaaaa aagctcaatgcattgtcttg tggaggatga 2820 ataaatgtga tcactgagac agccacttgg tttttggctctttcagagag gtctcaggtt 2880 ctttccatgc agactcctca gatc 2904 45 1227 DNAHomo sapiens 45 tggtccccta tcgcctccgc ctagcagctg ccatcggtgc gcccccacagctctaggacc 60 aataggcagg ccctagtgct gggactcgaa cggctattgg ttggccgagccgtggtgaga 120 gatggtgcgg tgcctgttct tggccctgca gagagctgtg ggcggttgttaaggcgaccg 180 ttcgtgacgt agcgccgtca ggccgagcag cccccaggcg attggctagacaatcgaacg 240 atcctctctt attggtcgaa ggctcgtcca gctccgagcg tgcgtaaggtgagggctcct 300 tccgctccgc gactgcgtta actggagcca ggctgagcgt cggcgccggggttcggtggc 360 ctctagtgag atctggaggt gaggcgggcg gtgaccgaga agaggggcaggggcggcggg 420 gagcggggcg agatgggtgg gagcggggtt tgggctgtgt tggtggcaattctggagctt 480 ccctcggccc tgggaagtgg ctaccggcag ctcctgcgga cctggagggggctgcggttg 540 cgctttgtcg gtgtggcagc tcggacccgc ggggactgca aggaatgtccttgaggcccg 600 gcaggccgag cggcggccgg catcagtgcc ggagtaaccc ggggtcccggggtgggcttg 660 agaggcgggc ggcggtctgg cctcttcgtg actgcggtca tcatcggtggacccgcgggg 720 cgtagctgcg ttcatcgtcc ctgttcagtc agagtaggca gtgctggctgcacggtcacg 780 aaaatcgggg cggaaagggt gtcaggcagg gtgacctcgg aggcccctggattcgagaaa 840 tgctaggggt ctatggggct gtcgggccgg cagctcgcag ggcagacgggagaagcgcct 900 gcatcccggg atccggcatt ctcgccagga actgctgttc gttagcacctttcttttagg 960 tgacgggaaa gatctctgta aatactgctg actaacttag aaccatgaaagaaccgtgga 1020 ttggtgtaga tgtgtctggt tatttacagg agaacggctt gagaggatgcggagcccaac 1080 gtgggacttc gcacaatgac tcaaaagatt cttctccctc tttttttttttttttttttg 1140 gtaaggggtg tagtctcctt ggtgctgata ttcttttagg aaaaatgtaccttggagata 1200 caaatataga acagttaatt tctgcag 1227 46 42 DNA ArtificialSequence Description of Artificial Sequence PCR primer 46 tgtaaaacgacggccagtag gaatactatt cacattccta tc 42 47 44 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 47 tgtaaaacga cggccagtctttgggcaatt cttaaatctt gtgc 44 48 30 DNA Artificial Sequence Descriptionof Artificial Sequence PCR primer 48 gcaaactcct gcctcaagtg atccgcctgc 3049 31 DNA Artificial Sequence Description of Artificial Sequence PCRprimer 49 gttcaagcag ttctcctgcc tcagcctcct g 31 50 22 DNA ArtificialSequence Description of Artificial Sequence dye terminator sequencingoligo 50 agaaaggtac acatactaca gg 22 51 25 DNA Artificial SequenceDescription of Artificial Sequence PCR primer 51 atgttgcagt gagccaagatcaagc 25 52 28 DNA Artificial Sequence Description of ArtificialSequence PCR primer 52 aacggatata aaggttgcgt ccagcagt 28 53 28 DNAArtificial Sequence Description of Artificial Sequence PCR primer 53aacggatata aaggttgcgt ccagctcc 28 54 291 DNA Homo sapiens 54 ttcacatttatttttctttt tatggtgtat tagtgcaagc ctgtctttgt attgtaaaat 60 ctaatgatacggtatttata ttatttttgt ttggcatttt ttgcattaaa tgaattattt 120 tgcagaggtatcttttaatt aaaaactaca gtgatttaat ttaaaaatta cattatttta 180 gcttagcattgtttgtatta aatggtttat aacatgaaat acagtccttc aagtcttctg 240 tttcatctctctctctgacc acaatttcat tttttttctc catttcttta g 291

1. A method for the diagnosis of a single nucleotide polymorphism inHMG-CoA reductase in a human, which method comprises determining thesequence of the nucleic acid of the human at at least one polymorphicposition selected from one or more of the following positions: position1962 in the coding sequence of the HMG-CoA reductase gene as defined bythe position in SEQ ID NO: 44, and/or positions 46 or 267 in thepromoter sequence of the HMG-CoA reductase gene as defined by thepositions in SEQ ID NO: 45; and/or position 129 in intron 2 as definedby the position in SEQ ID NO:20, and/or position 550 in intron 5 asdefined by the position in SEQ ID NO: 24, and/or position 37 in intron15 as defined by the position in SEQ ID NO:37, and/or position 345 inintron 18 as defined by the position in SEQ ID NO:40 of the HMG-CoAreductase gene, and determining the status of the human by reference topolymorphism in the HMG-CoA reductase gene.
 2. A method according toclaim 1 in which the polymorphism is further defined as the following:the single nucleotide polymorphism at position 1962 of the codingsequence is presence of A and/or G; the single nucleotide polymorphismat position 46 of the promoter is presence of T and/or C. the singlenucleotide polymorphism at position 267 of the promoter is presence of Cand/or G; the single nucleotide polymorphism at position 129 of intron 2is the presence or absence of an insertion of AA; the single nucleotidepolymorphism at position 550 of intron 5 is presence of T and/or A; thesingle nucleotide polymorphism at position 37 of intron 15 is presenceof A and/or G; and the single nucleotide polymorphism at position 345 ofintron 18 is presence of T and/or C.
 3. A method according to claim 1comprising determining the sequence of the nucleic acid of the human atposition 1962 in the coding sequence of the HMG-CoA reductase gene asdefined by the position in SEQ ID NO: 44 for presence of A and/or G. 4.A method according to claim 2 in which the sequence is determined by amethod selected from amplification refractory mutation system andrestriction fragment length polymorphism.
 5. Use of a method as definedin claim 2 to assess the pharmacogenetics of therapeutic compounds inthe treatment of HMG-CoA reductase mediated diseases.
 6. An isolatedpolynucleotide comprising at least 20 bases of the human HMG-CoAreductase gene and comprising a polymorphism selected from any one ofthe following: Region SEQ ID Position Polymorphism Exon 15 SEQ ID NO: 441962 A → G promoter SEQ ID NO: 45 46 C → G promoter SEQ ID NO: 45 267 T→ C Intron 2 SEQ ID NO: 20 129 CT → CAAT Intron 5 SEQ ID NO: 24 550 T →A Intron 15 SEQ ID NO: 37 37 A → G Intron 18 SEQ ID NO: 40 345 T → C


7. An allele specific primer or an allele specific oligonucleotide probecapable of detecting a HMG-CoA reductase gene polymorphism at one of thepositions defined in the table of claim
 6. 8. Use of any polymorphism asdefined in the table of claim 6 as a genetic marker in linkage studies.9 A computer readable medium comprising at least one polymorphism asdefined in the table of claim 6 stored on the medium. 10 A method oftreating a human in need of treatment with a HMG-CoA reductase inhibitordrug in which the method comprises: i) diagnosis of a single nucleotidepolymorphism in HMG-CoA reductase gene in the human, which diagnosiscomprises determining the sequence of the nucleic acid at one or more ofthe following positions: position 1962 in the coding sequence of theHMG-CoA reductase gene as defined by the position in SEQ ID NO: 44,and/or positions 46 or 267 in the promoter sequence of the HMG-CoAreductase gene as defined by the positions in SEQ ID NO: 45; and/orposition 129 in intron 2 as defined by the position in SEQ ID NO:20,and/or position 550 in intron 5 as defined by the position in SEQ ID NO:24, and/or position 37 in intron 15 as defined by the position in SEQ IDNO:37, and/or position 345 in intron 18 as defined by the position inSEQ ID NO:40 of the HMG-CoA reductase gene, and determining the statusof the human by reference to polymorphism in the HMG-CoA reductase gene;and ii) administering an effective amount of a HMG-CoA reductaseinhibitor. 11 An allelic variant of human HMG-CoA reductase polypeptidecomprising a valine at position 638 or a fragment thereof comprising atleast 10 amino acids provided that the fragment comprises the valine atposition
 638. 12 A polynucleotide sequence comprising any one of theintron sequences of HMG-CoA reductase defined in any one of SEQ ID NOS:18-41 and 54 or a complementary strand thereof or a sequence at least90% homologous thereto.